Anti-Tau (phospho S396) antibody [EPR2731]

11 product images

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  Image courtesy of Carl Hobbs, Kings College London, U.K.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)

  IHC image of Tau (phospho S396) staining in human Alzheimer hippocampus formalin fixed paraffin embedded tissue section. The section was pre-treated using heat mediated antigen retrieval with citrate buffer. The section was then incubated with unpurified ab109390 at 1/1000 dilution for 2 hoursat 21°C. A biotin conjugated goat-anti-rabbit antibody was used as a secondary at 1/250. The section shows clear neurofibrillary tangles in a subset of neurons.

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  Immunohistochemistry (Frozen sections) - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)

  Immunohistochemistry analysis of frozen mouse cerebrum tissue sections labeling Tau (phospho S396) with ab109390 at 1/100 (1 μg/mL). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit at 1/1000 (2 μg/mL) was used as the secondary antibody. Sections were fixed with 4% PFA and permeabilised with 0.2% Triton X-100. DAPI (blue) was used as nuclear counterstain. Antigen retrieval was heat mediated using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).
  

  Cytoplasmic staining on mouse cerebrum, the signal decreased after phosphatase treatment at 37°C for 2h.

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  Immunoprecipitation - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)

  ab109390 at 1/20 immunoprecipitating Tau (phospho S396) in Human brain lysate.
  

  Lane 1 (input): Human brain lysate (10μg)

  Lane 2 (+): ab109390 + Human brain lysate.

  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab109390 in Human brain lysate.

  For western blotting, ab109390 at 1/1000 dilution followed by VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/5000 dilution.

  Diluting / Blocking buffer and concentration: 5% NFDM/TBST.

  All lanes: Immunoprecipitation - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)

  Predicted band size: 78 kDa

  Observed band size: 50-70 kDa

  Exposure time: 3min

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  Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)

  Blocking/Diluting buffer and concentration 5% NFDM/TBST
  

  Tau assembles into oligomers as described in PMID: 28382304, 32692785 and 30120733.

  All lanes: Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390) at 1/1000 dilution

  Lane 1: Human brain lysate at 15 µg

  Lane 2: Human brain lysates and the membrane was incubated with alkaline phosphatase at 15 µg

  Lane 3: Human brain lysates and the membrane was incubated with lambda phosphatase at 15 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Predicted band size: 78 kDa

  Observed band size: 50-79 kDa

  Exposure time: 100s

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  Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)

  Blocking buffer: 5% NFDM/TBST

  Dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390) at 1/20000 dilution

  Lane 1: Untreated SH-SY5Y at 10 µg

  Lane 2: SH-SY5Y treated with alkaline phosphatase at 10 µg

  Secondary

  All lanes: HRP goat ant-rabbit (H+L) at 1/1000 dilution

  Predicted band size: 78 kDa

  Observed band size: 50-70 kDa

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  Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)

  Diluting/Diluting buffer and concentration 5% NFDM/TBST
  

  Tau assembles into oligomers as described in PMID: 28382304, 32692785 and 30120733..

  All lanes: Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390) at 1/1000 dilution

  Lane 1: Mouse brain lysate at 15 µg

  Lane 2: Mouse brain lysates and the membrane was incubated with alkaline phosphatase at 15 µg

  Lane 3: Mouse brain lysates and the membrane was incubated with lambda phosphatase at 15 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Predicted band size: 78 kDa

  Observed band size: 50-79 kDa

  Exposure time: 10s

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  Immunohistochemistry (Frozen sections) - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)

  IHC image of Tau (phospho S396) staining in a section of frozen normal human Alzheimer brain performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab109390, 1/1000 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.
  

  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)

  Immunohistochemical staining of paraffin embedded human glioblastoma with purified ab109390 at a dilution of 1/4000. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained wirh hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

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  Immunohistochemistry (Frozen sections) - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)
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  Dot Blot - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)

  Dot blot analysis of Tau (phospho S396) phospho peptide (Lane 1) and Tau non-phospho peptide (Lane 2) labeling Tau (phospho S396) with ab109390 at a dilution of 1/1000. Goat Anti-Rabbit IgG H&L (HRP) ab97051 (Peroxidase conjugated goat anti-rabbit IgG) (H+L) at 1/100 000 was used as the secondary antibody.
  

  Blocking and diluting buffer: 5% NFDM/TBST.

  Exposure time: 3 minutes.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390)

  Immunohistochemistry analysis of paraffin-embedded human colon tissue sections labelling Tau (phospho S396) with ab109390 at 1/4000 dilution (0.026 μg/mL). The section was incubated with ab109390 for 30 mins at room temperature. Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101) was used as the secondary antibody. Sections were counterstained with Hematoxylin. Antigen retrieval was heat mediated using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes.
  Positive staining on ganglions of human colon without alkaline phosphatase treatment (image A); No signal was detected when tissues were treated with alkaline phosphatase (image B).The immunostaining was performed on a Leica Biosystems BOND® RX instrument.